Project Summary NAFLD affects approximately 34% of obese children ages 3-18 in North America and half have already progressed to the more severe nonalcoholic steatohepatitis (NASH) at time of diagnosis. Maternal obesity (OB), excess gestational weight gain (GWG), and gestational diabetes mellitus (GDM) are associated with increased risk for childhood OB and NAFLD, but the driver of this association is unclear. We, and others, have found that maternal obesity in humans in combination with GDM or a maternal high-fat diet (HFD) in Non-Human Primates (NHP) independent of obesity, results in increased intrahepatic fat in infants and gut dysbiosis in NHP. Hepatic steatosis and dybiosis persists at 1 yr in juvenile NHP despite switching the offspring to a normal diet at weaning. Remarkably, hepatic macrophages (M?) in juvenile NHP offspring of OB mothers fed a HFD during pregnancy are programmed for inflammation, even if the offspring are switched to a normal diet. Many studies have linked gut dybiosis to NAFLD, however evidence that early changes in the infant gut MB precede disease is lacking. Pilot studies from our ADA sponsored longitudinal mother/infant studies show that when microbes from 2 wk old infants born to OB mothers (Inf-OB) are transferred to germ-free (GF) mice, the mice exhibit increased liver fat, recruitment of pro-inflammatory liver M?, and histological evidence for early fibrosis. To isolate the effect of maternal diet, we are also collecting stool samples from an NIH-sponsored RCT in GDM women randomized to either a conventional higher fat (45%)/low carb (40%) diet or a low fat (25%)/higher complex carb (60%) diet? all meals provided, for which pilot data suggests lower infant adiposity. Whether maternal diet is capable of independently altering the infant MB to increase the susceptibility to NAFLD or whether maternal phenotype is a more important driver is completely unknown. Because early infant immune development is highly dependent on triggers provided by the MB, our central hypothesis is that an imbalance in gut microbes in infants born to OB mothers or NW mothers with excess GWG triggers remodeling of pro-inflammatory liver M? leading to increased NAFLD risk, and that a HFD further exacerbates this risk. We will discern the mechanisms by which infant dysiosis promotes liver and bone marrow-derived M? metabolic re-programming, inflammation, and progression to NASH in GF mice, and the time course using infant stool samples at key developmental time points. We hypothesize that infants born to mothers with OB, excess GWG or GDM on HFD will have pathologic levels of metabolic and inflammatory markers compared to NW, including BA and SCFA that correlate with specific gut bacteria and NAFLD in GF mice. We will define the functional relevance of early infant microbial communities to promote NAFLD according to maternal OB, excess GWG, and GDM, and whether a nutritional intervention can promote a healthier metabolic microbiome profile. Chronic low-grade inflammation is strongly associated with OB, NAFLD and CVD, thus the implications of this study are wide-reaching and important for understanding the causative role of early microbes and developing realistic therapeutic strategies.